Identifying the enzyme involved in Vacuolar ATPase acetylation during doxorubicin-induced cardiotoxicity

Doxorubicin is an established anticancer medication infamous for its bright colouration and extremely toxic side effects. Emerging studies support that the imbalance between acetylation and deacetylation disrupts the autophagic flux leading to doxorubicin-induced cardiotoxicity. Vacuolar ATPases are a family of electrogenic proton pumps present on the lysosomal membrane that create an acidic environment for proteases to degrade proteins. Our preliminary study found that acetylated Vacuolar ATPase subunit V0 D1 levels increased in doxorubicin-injected mouse hearts. However, it is unknown how acetylation of subunit V0 D1 is modulated and whether this modification plays a role in doxorubicin-induced cardiotoxicity. The focus of the project is to identify the specific acetylase involved in acetylating Vacuolar ATPase subunit V0 D1 using histone acetyltransferase (HAT) inhibitors and its potential role in doxorubicin-induced cardiotoxicity. For example, Garcinol is a p300 HAT inhibitor derived from a plant called Garcinia Indica and past studies revealed that Garcinol natural properties have cardioprotective effects. Cardiomyocytes will be used as a model and cell viability, apoptosis, lactate dehydrogenase release, autophagic flux, Vacuolar ATPase activity (lysosomal pH), and Vacuolar ATPase subunit V0 D1 acetylation will be analyzed. It is hypothesized that selective HAT inhibition will prevent Vacuolar ATPase subunit V0 D1 hyperacetylation and allow for regulated autophagic flux to restore cell viability during doxorubicin-induced cardiotoxicity

Corrigendum: Calpain activation contributes to hyperglycemia-induced apoptosis in cardiomyocytes (Cardiovascular Research (2009) 84 (100-110) DOI: 10.1093/cvr/cvp189)

The authors regret that they mixed up the immunoblot images in the above article, and mistakenly placed the wrong images for Fig. 1 C1 and C2. Please find the corrected Fig. 1 C1 and C2 below. (Figure Presented)

Targeted inhibition of calpain reduces myocardial hypertrophy and fibrosis in mouse models of type 1 diabetes

OBJECTIVE - Recently we have shown that calpain-1 activation contributes to cardiomyocyte apoptosis induced by hyperglycemia. This study was undertaken to investigate whether targeted disruption of calpain would reduce myocardial hypertrophy and fibrosis in mouse models of type 1 diabetes. RESEARCH DESIGN AND METHODS - Diabetes in mice was induced by injection of streptozotocin (STZ), and OVE26 mice were also used as a type 1 diabetic model. The function of calpain was genetically manipulated by cardiomyocyte-specific knockout Capn4 in mice and the use of calpastatin transgenic mice. Myocardial hypertrophy and fibrosis were investigated 2 and 5 months after STZ injection or in OVE26 diabetic mice at the age of 5 months. Cultured isolated adult mouse cardiac fibroblast cells were also investigated under high glucose conditions. RESULTS - Calpain activity, cardiomyocyte cross-sectional areas, and myocardial collagen deposition were significantly increased in both STZ-induced and OVE26 diabetic hearts, and these were accompanied by elevated expression of hypertrophic and fibrotic collagen genes. Deficiency of Capn4 or overexpression of calpastatin reduced myocardial hypertrophy and fibrosis in both diabetic models, leading to the improvement of myocardial function. These effects were associated with a normalization of the nuclear factor of activated T-cell nuclear factor-kB and matrix metalloproteinase (MMP) activities in diabetic hearts. In cultured cardiac fibroblasts, high glucose-induced proliferation and MMP activities were prevented by calpain inhibition. CONCLUSIONS - Myocardial hypertrophy and fibrosis in diabetic mice are attenuated by reduction of calpain function. Thus targeted inhibition of calpain represents a potential novel therapeutic strategy for reversing diabetic cardiomyopathy. © 2011 by the American Diabetes Association

Cortico-amygdalar connectivity and externalizing/internalizing behavior in children with neurodevelopmental disorders

Background: Externalizing and internalizing behaviors contribute to clinical impairment in children with neurodevelopmental disorders (NDDs). Although associations between externalizing or internalizing behaviors and cortico-amygdalar connectivity have been found in clinical and non-clinical pediatric samples, no previous study has examined whether similar shared associations are present across children with different NDDs. Methods: Multi-modal neuroimaging and behavioral data from the Province of Ontario Neurodevelopmental Disorders (POND) Network were used. POND participants aged 6–18 years with a primary diagnosis of autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD) or obsessive–compulsive disorder (OCD), as well as typically developing children (TDC) with T1-weighted, resting-state fMRI or diffusion weighted imaging (DWI) and parent-report Child Behavioral Checklist (CBCL) data available, were analyzed (total n = 346). Associations between externalizing or internalizing behavior and cortico-amygdalar structural and functional connectivity indices were examined using linear regressions, controlling for age, gender, and image-modality specific covariates. Behavior-by-diagnosis interaction effects were also examined. Results: No significant linear associations (or diagnosis-by-behavior interaction effects) were found between CBCL-measured externalizing or internalizing behaviors and any of the connectivity indices examined. Post-hoc bootstrapping analyses indicated stability and reliability of these null results. Conclusions: The current study provides evidence towards an absence of a shared linear relationship between internalizing or externalizing behaviors and cortico-amygdalar connectivity properties across a transdiagnostic sample of children with different primary NDD diagnoses and TDC. Different methodological approaches, including incorporation of multi-dimensional behavioral data (e.g., task-based fMRI) or clustering approaches may be needed to clarify complex brain-behavior relationships relevant to externalizing/internalizing behaviors in heterogeneous clinical NDD populations

Prevention of hyperglycemia-induced myocardial apoptosis by gene silencing of Toll-like receptor-4

<p>Abstract</p> <p>Background</p> <p>Apoptosis is an early event involved in cardiomyopathy associated with diabetes mellitus. Toll-like receptor (TLR) signaling triggers cell apoptosis through multiple mechanisms. Up-regulation of TLR4 expression has been shown in diabetic mice. This study aimed to delineate the role of TLR4 in myocardial apoptosis, and to block this process through gene silencing of TLR4 in the myocardia of diabetic mice.</p> <p>Methods</p> <p>Diabetes was induced in C57/BL6 mice by the injection of streptozotocin. Diabetic mice were treated with 50 μg of TLR4 siRNA or scrambled siRNA as control. Myocardial apoptosis was determined by TUNEL assay.</p> <p>Results</p> <p>After 7 days of hyperglycemia, the level of TLR4 mRNA in myocardial tissue was significantly elevated. Treatment of TLR4 siRNA knocked down gene expression as well as diminished its elevation in diabetic mice. Apoptosis was evident in cardiac tissues of diabetic mice as detected by a TUNEL assay. In contrast, treatment with TLR4 siRNA minimized apoptosis in myocardial tissues. Mechanistically, caspase-3 activation was significantly inhibited in mice that were treated with TLR4 siRNA, but not in mice treated with control siRNA. Additionally, gene silencing of TLR4 resulted in suppression of apoptotic cascades, such as Fas and caspase-3 gene expression. TLR4 deficiency resulted in inhibition of reactive oxygen species (ROS) production and NADPH oxidase activity, suggesting suppression of hyperglycemia-induced apoptosis by TLR4 is associated with attenuation of oxidative stress to the cardiomyocytes.</p> <p>Conclusions</p> <p>In summary, we present novel evidence that TLR4 plays a critical role in cardiac apoptosis. This is the first demonstration of the prevention of cardiac apoptosis in diabetic mice through silencing of the TLR4 gene.</p

Cardiac Transcription Factor Nkx2.5 Is Downregulated under Excessive O-GlcNAcylation Condition

Post-translational modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) is linked the development of diabetic cardiomyopathy. We investigated whether Nkx2.5 protein, a cardiac transcription factor, is regulated by O-GlcNAc. Recombinant Nkx2.5 (myc-Nkx2.5) proteins were reduced by treatment with the O-GlcNAcase inhibitors STZ and O-(2-acetamido-2-deoxy-D-glucopyroanosylidene)-amino-N-phenylcarbamate; PUGNAC) as well as the overexpression of recombinant O-GlcNAc transferase (OGT-flag). Co-immunoprecipitation analysis revealed that myc-Nkx2.5 and OGT-flag proteins interacted and myc-Nkx2.5 proteins were modified by O-GlcNAc. In addition, Nkx2.5 proteins were reduced in the heart tissue of streptozotocin (STZ)-induced diabetic mice and O-GlcNAc modification of Nkx2.5 protein increased in diabetic heart tissue compared with non-diabetic heart. Thus, excessive O-GlcNAcylation causes downregulation of Nkx2.5, which may be an underlying contributing factor for the development of diabetic cardiomyopathy

Hsp20 Functions as a Novel Cardiokine in Promoting Angiogenesis via Activation of VEGFR2

Heat shock proteins (Hsps) are well appreciated as intrinsic protectors of cardiomyocytes against numerous stresses. Recent studies have indicated that Hsp20 (HspB6), a small heat shock protein, was increased in blood from cardiomyopathic hamsters. However, the exact source of the increased circulating Hsp20 and its potential role remain obscure. In this study, we observed that the circulating Hsp20 was increased in a transgenic mouse model with cardiac-specific overexpression of Hsp20, compared with wild-type mice, suggesting its origin from cardiomyocytes. Consistently, culture media harvested from Hsp20-overexpressing cardiomyocytes by Ad.Hsp20 infection contained an increased amount of Hsp20, compared to control media. Furthermore, we identified that Hsp20 was secreted through exosomes, independent of the endoplasmic reticulum-Golgi pathway. To investigate whether extracellular Hsp20 promotes angiogenesis, we treated human umbilical vein endothelial cells (HUVECs) with recombinant human Hsp20 protein, and observed that Hsp20 dose-dependently promoted HUVEC proliferation, migration and tube formation. Moreover, a protein binding assay and immunostaining revealed an interaction between Hsp20 and VEGFR2. Accordingly, stimulatory effects of Hsp20 on HUVECs were blocked by a VEGFR2 neutralizing antibody and CBO-P11 (a VEGFR inhibitor). These in vitro data are consistent with the in vivo findings that capillary density was significantly enhanced in Hsp20-overexpressing hearts, compared to non-transgenic hearts. Collectively, our findings demonstrate that Hsp20 serves as a novel cardiokine in regulating myocardial angiogenesis through activation of the VEGFR signaling cascade

Thaliporphine Preserves Cardiac Function of Endotoxemic Rabbits by Both Directly and Indirectly Attenuating NFκB Signaling Pathway

Cardiac depression in sepsis is associated with the increased morbidity and mortality. Although myofilaments damage, autonomic dysfunction, and apoptosis play roles in sepsis-induced myocardial dysfunction, the underlying mechanism is not clear. All of these possible factors are related to NFκB signaling, which plays the main role in sepsis signaling. Thaliporphine was determined to possess anti-inflammatory and cardioprotective activity by suppressing NFκB signaling in rodents. The purpose of this study is to further prove this protective effect in larger septic animals, and try to find the underlying mechanisms. The systolic and diastolic functions were evaluated in vivo by pressure-volume analysis at different preloads. Both preload-dependent and -independent hemodynamic parameters were performed. Inflammatory factors of whole blood and serum samples were analyzed. Several sepsis-related signaling pathways were also determined at protein level. Changes detected by conductance catheter showed Thaliporphine could recover impaired left ventricular systolic function after 4 hours LPS injection. It could also reverse the LPS induced steeper EDPVR and gentler ESPVR, thus improve Ees, Ea, and PRSW. Thaliporphine may exert this protective effect by decreasing TNFα and caspase3 dependent cell apoptosis, which was consistent with the decreased serum cTnI and LDH concentration. Thaliporphine could protect sepsis-associated myocardial dysfunction in both preload-dependent and -independent ways. It may exert these protective effects by both increase of “good”-PI3K/Akt/mTOR and decrease of “bad”-p38/NFκB pathways, which followed by diminishing TNFα and caspase3 dependent cell apoptosis